Next-generation sequencing technologies have revolutionized genomics in their ability to generate enormous amounts of sequence read-outs in a single run. However, accurate quantification of the input DNA library is critical in order to assure the optimal performance of the sequencing instrument and to reduce overall costs.
“Sequencing of DNA libraries can take several days to run, so it is important to obtain the most information you can during a run,” Bernd Buehler, Ph.D., senior scientist at Agilent Technologies, says. “For example, on the Illumina Genome Analyzer, the cDNA library is immobilized and amplified on a chip in a process called cluster generation. If the amount of DNA loaded is too high, the DNA clusters could overlap and produce poor quality data. On the other hand, loading too low of an amount will result in low cluster density and reduce overall efficiency.”
Dr. Buehler says that using qPCR can improve both the accuracy and efficiency of the process. “We have done studies looking at the correlation of qPCR results with our High Sensitivity DNA Quantification Kit on the 2100 Bioanalyzer. In all cases, qPCR proved to be a complementary method for quantifying high-quality library preparations.”
Additionally, qPCR provides several other advantages, Dr. Buehler says. “qPCR is very specific and only detects adapter-containing sequences since the identical primer sequences are used for library amplification. Also qPCR is exquisitely sensitive and can be used to help avoid amplification bias by reducing or eliminating the need to amplify DNA before sequencing.”
However, utilizing an accurate standard is critical. “Principally, two sources of a standard can be used for quantification, such as a previously quantified DNA library that was successfully sequenced, or a plasmid standard. The latter has the advantage since the standard can be easily reproduced for future use.”
“qPCR assays may provide an ideal solution for quantification of sequencing libraries,” Dr. Buehler says. “We feel that more and more researchers will be using this strategy in the future.”
GE Global Research is developing a third-generation sequencing technology that aims to be less expensive and more accurate. “We are working on a means to interrogate DNA that can be used for high-throughput, single-molecule DNA sequencing,” John Nelson, Ph.D., molecular biologist, reports. “This method can utilize less expensive optics by sequencing clonally amplified DNA, or alternatively, can be developed for a high-end system that can interrogate single molecules.”
The proposed method is a sequencing-by-synthesis strategy. “This uses two separate innovations together to accomplish DNA sequencing. First, we had already developed terminal phosphate labeled nucleotides with a dye attached that incorporates and is then removed from the growing DNA strand. Next, we developed a method to ‘freeze’ the DNA polymerase as it is incorporated into this unique nucleotide.
“In this state, the three-part complex of DNA strand, polymerase, and incoming base are quite stable, and can be washed and interrogated to determine the identify of the trapped base by the color of the dye in the complex. After interrogation we allow the polymerase to add that single base and proceed on to form the complex on the next template base. This cycle is repeated over and over.”
According to Dr. Nelson, the new method has several advantages. “For instance, our method can be used to step DNA synthesis even through homopolymers, reducing some of the issues associated with blocks of identical bases. The method can also be used for single-molecule sequencing and provides a stable signal that can be interrogated carefully but requires no chemistry besides DNA polymerization to completely remove the label used. Since we add a new enzyme for each step, enzyme stability is not an issue.”
GE Global Research has demonstrated proof-of-concept for the method. It is now optimizing various aspects of its new sequencing system.